ethyl N2-dodecanoyl-.sc.l.sc.-argininate hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key study: Test method according to OECD 471. GLP study. LAE did not show mutagenic potential in S. typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA/pKM101 with and without metabolic activation.

Key study: Test method according to OECD 476 - GLP study. LAE did not demonstrate mutagenic potential in an in vitro mouse lymphoma assay with and without metabolic activation.

Key study: Test method according to OECD 473. GLP study. LAE was not clastogenic in an in vitro chromosome aberration assay with and without metabolic activation in human lymphocytes.

Key study: Test method according to OECD 471. GLP study. LAE (25 %) did not show mutagenic potential in S. typhimurium strains TA1535, TA1537, TA98, TA100 with and without metabolic activation.

Key study: Test method according to OECD 476 - GLP study. LAE (25 %) did not demonstrate mutagenic potential in an in vitro mouse lymphoma assay with and without metabolic activation.

Key study: Test method according to OECD 473. GLP study. LAE (25 %) was not clastogenic in an in vitro chromosome aberration assay with and without metabolic activation in human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 18, 1995 to August 24, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rats

Test concentrations with justification for top dose:

TEST 1: Preliminary test
Concentration range (with metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Concentration range (without metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

TEST 2: Mutation test
Concentration range (with metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Concentration range (without metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

Solvent: Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

Without methabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA1535; TA100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

other: 2-Aminoanthracene

Remarks:

All strains

Details on test system and experimental conditions:

METHOD OF APPLICATION:

TEST 1: Preliminary test: in agar (plate incorporation)
An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. A single petri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background bacterial lawn was examined. Revertant colonies were counted using a Seescan Automatic Colony Counter.

NUMBER OF REPLICATIONS: 3


TEST 2: MAIN TEST: in agar (plate incorporation)
An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 /tg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period revertant colonies were counted using a Seescan Automatic Colony Counter.


NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The mean number of revertant colonies for each treatment group was compared with those obtained for the solvent/vehicle control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, the test substance will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.
b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent/vehicle controls in either mutation test, the test substance will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs a) and b), even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at a maximum exposure to LAE at 5000 and 500 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Concentrations of test substance up to 5000 µg/plate were tested in the first range-finder test.
The range-finder test was repeated using lower concentrations, up to 150 µg/plate, because of observed toxicity. Both tests were standard plate incorporation assays.
A second main test was conducted at concentrations up to 150 µg/plate. This test involved a pre-incubation stage.

RANGE-FINDING/SCREENING STUDIES:
The revertant colony counts obtained in the preliminary toxicity test was observed. The test substance was toxic at the highest concentration towards tester strains TA 98 and TA 100, and towards TA 1537 in the presence of S-9 mix only. Toxicity was also seen towards TA 98 at 500 µg/plate in the absence of S-9 mix. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests followed by a further five dose levels.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Following treatment with the test substance in the first mutation test, toxicity was observed towards all the strains between 5000 and 500 µg/plate, except TA 1535 at 500 µg/plate in the presence of S-9 mix. Therefore the range of dose levels selected was changed, and the highest concentration was reduced to 500 µg/plate.

Conclusions:

The in vitro assessment of the mutagenic potential of the test substance in a bacterial system performed according to OECD Guideline 471 of Genetic Toxicology on Bacterial Reverse Mutation Test. The test substance showed no evidence of mutagenic activity observed in the Bacterial reverse Mutation Test with and without metabolic activation tested for strains of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100).

Executive summary:

This report describes a study designed to assess the mutagenic potential of the test substance in a bacterial system. The study was conducted in compliance with the following guidelines: OECD Guidelines for the Testing of Chemicals (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471.

In this in vitro assessment of the mutagenic potential of the test substance, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test substance, diluted in water which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary toxicity test with dose levels of up to 5000 µg/plate toxicity was observed at the highest concentration towards TA 98 and TA 100, and towards TA 1537 in the presence of S-9 mix only. Toxicity was also seen towards TA 98 at 500 µg/plate in the absence of S-9 mix. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50, 15, 5 µg/plate.

Following treatment with the test substance in the first mutation test, toxicity was observed towards all the strains between 5000 and 500 µg/plate, except TA 1535 at 500/µg/plate in the presence of S-9 mix. Therefore the range of dose levels selected was changed, and the highest concentration was reduced to 500 µg/plate.

Following treatment with the test substance in the second mutation test, toxicity was observed towards all the strains at 500 µg/plate, except TA 1535 in the presence of S-9 mix.

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with the test substance at any dose level, in the presence or absence of S-9 mix, in either mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparation.

It is concluded that, when tested in water, the test substance was not mutagenic in this bacterial system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 18, 2001 to March 29, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture, Forestry and Fisheries. (1985) Notification of Director General, Agricultural Production Bureau. NohSan No. 4200.

Qualifier:

according to guideline

Guideline:

other: Joint Directives of J EPA, J MHW and J MITI. (31 October 1997) Kanpoan No. 287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

TEST 1: Range-finding test
Concentration range (with metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Concentration range (without metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

TEST 1: Range-finding Repeat test
Concentration range (with metabolic activation): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
Concentration range (without metabolic activation): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate

TEST 2: Main test
Concentration range (with metabolic activation): 1.5, 5, 15, 50, 150 µg/plate
Concentration range (without metabolic activation): 1.5, 5, 15, 50, 150 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

other: 2-Aminoanthracene

Remarks:

TA1535; E. coli WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

TA98; TA100; TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without methabolic activation

Positive control substance:

sodium azide

Remarks:

TA1535; TA100 Migrated to IUCLID6: Sodium azide; 9-Aminoacridine; 2-Nitrofluorene; 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without methabolic activation

Positive control substance:

9-aminoacridine

Remarks:

TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without methabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without methabolic activation

Positive control substance:

other: 2-(2-FuryI)-3-(5-nitro-2-fwyl) acrylamide (AF-2)

Remarks:

E. coli WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION:

TEST 1: Range finding: in agar (plate incorporation)
An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 M sodium phosphate buffer (pH 7.4) were placed in glass tubes. An aliquot of 0.1 ml of the test dilution, positive or negative control was added, followed immediately by 2 ml of molten agar containing 0.5mM histidinefoiotin/tryptophan. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Three petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at 37°C for c.a. 72 hours. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.
NUMBER OF REPLICATIONS: 3


TEST 1 Repeat: Range finding Repeat: in agar (plate incorporation)
The same method as previous test
NUMBER OF REPLICATIONS: 3


TEST 2: MAIN TEST: Pre-incubation assay
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. 150 µg/plate was again chosen as the top concentration, but only five concentrations were used.

Evaluation criteria:

The mean number of revertant colonies for each treatment group was compared with those obtained for the solvent/vehicle control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, the test substance will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.
b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent/vehicle controls in either mutation test, the test substance will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs a) and b), even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In all strains at 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Concentrations of test substance up to 5000 µg/plate were tested in the first range-finder test.
The range-finder test was repeated using lower concentrations, up to 150 µg/plate, because of observed toxicity. Both tests were standard plate incorporation assays.
A second main test was conducted at concentrations up to 150 µg/plate. This test involved a pre-incubation stage.

All concentrations cited in this report are expressed in terms of pure LAE, i.e. correction was made for the purity of 93.2 %.

Conclusions:

According to OECD Guideline 471 and EU method, the in vitro assessment of the mutagenic potential of Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) in a bacterial system performed showed no evidence of mutagenic activity observed in the Bacterial reverse Mutation Test with and without metabolic activation.

Executive summary:

This report describes a study designed to assess the mutagenic potential of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) in a bacterial system. The study was conducted in compliance with the following guidelines: OECD Guidelines for the Testing of Chemicals (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471; EEC (1992) Part B: Methods for Determination of Toxicity, B.13. Other effects - Mutagenicity: Escherichia coli- Reverse Mutation Assay and EEC (1992) Part B: Methods for Determination of Toxicity, B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay.

In this in vitro assessment of the mutagenic potential of LAE, histidine dependent auxotrophic mutants of Salmonella typhimurium,strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to the test substance diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a negative control.

 

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). The first (range-finding) was a standard plate incorporation assay; the second involved a pre-incubation stage. The absence of colonies on sterility check plates confirmed the absence of microbial contamination.

Concentrations of LAE up to 5000 µg/plate were tested in the first mutation test. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The first test was repeated using lower LAE concentrations, up to 150 µg/plate, because of observed toxicity.

The same maximum concentration was selected for the second test. Other concentrations used were a series of c.a. half-log₁₀dilutions of the highest concentration. Toxicity (observed as thinning of the background lawn of non-revertant cells, together with a reduction in revertant colony numbers) was seen in all strains following exposure to LAE at 150 µg/plate in the repeated first test and in the second test.

 

No evidence of mutagenic activity was seen at any concentration of LAE in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, LAE showed no evidence of mutagenic activity in this bacterial system. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 8, 2004 to May 24, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals; Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: R0 (RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM
L-glutamine and 50 ug/mL gentamycin); R10p (R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v);
R30p (R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes.

Additional strain / cell type characteristics:

other: Heterozygous at the thymidine kinase locus

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate derived from Aroclor 1254 induced rats (S-9 mix).

Test concentrations with justification for top dose:

Concentration range in the main test (without metabolic activation): -S9 mix (3 hours)
- Test 1 10, 24, 28, 30, 34, 38, 40, 45 and 50 µg/mL
- Test 2 10, 24, 26, 28, 30, 31, 32, 33 and 34 µg/mL

Concentration range in the main test (with metabolic activation): +S9 mix (3 hours)
- Test 1 10, 40, 42, 43, 44, 45, 46, 47, 48 and 50 µg/mL
- Test 2 15, 30, 42, 43, 43.5, 44, 44.5, 45, 45.5 and 46 µg/mL

Concentration range in the main test (without metabolic activation): -S9 mix (24 hours)
- Test 3 1, 10, 20, 30, 32, 34, 36, 38, 40 and 45 µg/mL
- Test 4 1, 20, 30, 40, 42.5, 45, 47.5 and 50 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: LAE was found to be soluble at approximately 236 mg/ml

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

FIRST AND SECOND MAIN MUTAGENICITY TESTS:
METHOD OF APPLICATION: Duplicate 6 mL aliquots of a suspension of 2.0 x 10E6 cells/mL had 4 mL of R0 or S9 mix added as appropriate. Quadruplicate cultures were prepared for solvent controls. Aliquots of 100 µL of test substance dilution (at 100 times the desired final concentration), solvent or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37ºC. Following the 3-hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After the plates had been incubated for approximately 10-14 days for mutant plates, the number of empty wells was assessed for each 96-well plate.

DURATION
- Exposure duration: 3 h
- Selection time (if incubation with a selection agent): 48 h

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 ug/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency was assessed by plating 1.6 cells/well, two plates being prepared per culture.

THIRD AND FOURTH MAIN MUTAGENICITY TESTS:
METHOD OF APPLICATION: Duplicate 10 mL cultures at 0.3 x 10E6 cells/mL were treated for 24 hours with 100 µL of test substance, solvent or positive control. Following this, the procedure was the same as in the 3-hour treatment.

DURATION
- Exposure duration: 24 h
- Selection time (if incubation with a selection agent): 48 h

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 ug/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency was assessed by plating 1.6 cells/well, two plates being prepared per culture.

Evaluation criteria:

The criteria used to assess whether an assay was valid are based on those published by Sofuni et al, 1997 and Moore et al, 2000 and Moore et al, 2003.

Statistics:

The statistical significance of the data was analysed by following the methods described by Robinson et al (1989).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at the highest concentrations)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
Some evidence of polyploidy-inducing activity was seen, but this mainly at cytotoxic dose levels and may not be of biological significance.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, a 3-hour exposure to LAE at 0.29-600 µg/mL in the absence and presence of S9 mix resulted in RSG of 105-0% and 114-0% respectively. A continuous exposure to 0.29-600 µg/mL for 24 hours in the absence of S9 mix resulted in RSG of 98-0%. Concentrations used in the main test were based upon these data.

Conclusions:

Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells according to OECD Guideline 476. Results did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

The test substance, N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) , was tested for potential mutagenicity in the mouse lymphoma L5178Y cell mutation test according to OECD Guideline 476. This test involves detection of mutation of mouse lymphoma L5178Y cells using the microtitre^Rfluctuation technique in 96-well microtitre plates. The cells are heterozygous at the thymidine kinase (tk) locus (TK^/-), and forward mutation (TK^-/-) is detected by the ability of these cells to divide and form colonies in the presence of trifluorothymidine (TFT), a toxic analogue of thymidine.

LAE was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+^/-) to the thymidine kinase deficient genotype (TK^-/-).

The study comprised a preliminary toxicity test and six independent mutagenicity tests. The cells were exposed for 3 or 24 hours in the absence of exogenous metabolic activation (S9 mix) and 3 hours in the presence of S9 mix.

LAE was found to be soluble at approximately 236 mg/mL in dimethyl sulphoxide (DMSO). Solutions at this concentration and at approximately 118 mg/mL, when dosed at 1% in medium, formed precipitate that settled out. Solutions of LAE at approximately 15 mg/mL, 30 mg/mL and 59 mg/mL formed cloudy/milky suspensions in medium. Solutions of approximately 7 mg/mL and lower did not form visible precipitate in medium. No colour change was observed at any of the concentrations. The maximum concentration tested in the preliminary toxicity test was 60 mg/mL (final concentration in medium of 600 ug/mL), the objective being to test up to and beyond the limit of solubility.

In the main tests in the absence of S9 mix with a 3-hour exposure, the maximum concentration assessed for determination of mutant frequency was 34 µg/mL. There were no statistically significant increases in mutant frequency at any of the concentrations tested at acceptable levels of toxicity.

In the main tests in the presenceof S9 mix with a 3-hour exposure, the maximum concentration assessed for determination of mutant frequency was 46 µg/mL. There were no statistically significant increases in mutant frequency at any of the concentrations tested at acceptable levels of toxicity.

In the main tests in the absence of S9 mix with a 24-hour exposure, the maximum concentration assessed for determination of mutant frequency was 50 µg/mL. There were no statistically significant increases in mutant frequency at any of the concentrations tested at acceptable levels of toxicity.

In all tests the concurrent solvent and positive control were within the ranges determined from the historical solvent and positive control data.

It was concluded that LAE did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 25, 2001 to June 13, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human blood was collected aseptically from healthy, non-smoking male donors.

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Male donor
- Sex, age and number of blood donors if applicable: Male non smoking donor

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 supplemeted with 10 % foetal calf serum, 1 unit/mL heparin, 20IU/mL penicillin /20 µg/mL streptomycin and 2.0 mM glutamine.
- Properly maintained: Yes.

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate derived from Aroclor 1254 induced rats (S-9 mix).

Test concentrations with justification for top dose:

FIRST TEST:
Concentration range (without metabolic activation): 50, 100 and 200 µg/ml Concentration range (with metabolic activation): 50, 100 and 200 µg/ml
SECOND TEST
Concentration range (with metabolic activation): 50, 100 and 150 µg/ml
Concentration range in the main test (without metabolic activation): 50, 100 and 150 µg/ml

Vehicle / solvent:

Dimethylsulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

FIRST TEST:
METHOD OF APPLICATION: Human blood was collected aseptically from healthy, non-smoking male donors, pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum. Aliquots of the cell suspension were placed in sterile universal containers and incubated at 37 ºC for approximately 48 hours. After 48 h, the different treatments were applied to the cells.

DURATION
- Exposure duration: 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 17 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: One hundred metaphase figures were examined, from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: The proportion of mitotic cells per 1000 cells in each culture was recorded.

OTHER EXAMINATIONS:
- Determination of polyploidy: Out of 500 metaphase cells was determined where possible
- Determination of endoreplication: observation and noted when seen.

SECOND TEST:
METHOD OF APPLICATION: Human blood was collected aseptically from healthy, non-smoking male donors, pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum. Aliquots of the cell suspension were placed in sterile universal containers and incubated at 37 ºC for approximately 48 hours. After 48 h, the different treatments were applied to the cells.

DURATION
- Exposure duration: 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours harvest time

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: One hundred metaphase figures were examined, from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: The proportion of mitotic cells per 1000 cells in each culture was recorded.

OTHER EXAMINATIONS:
- Determination of polyploidy: Out of 500 metaphase cells was determined where possible
- Determination of endoreplication: observation and noted when seen.

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The increases exceed the negative control range, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.

Key result

Species / strain:

lymphocytes: Human blood was collected aseptically from healthy, non-smoking male donors.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 200 and 150 µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Some evidence of polyploidy-inducing activity was seen, but this mainly at cytotoxic dose levels and may not be of biological significance.

Conclusions:

Nα-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) was tested for mutagenic potential in an in vitro mammalian cell mutation assay according to OECD Guideline 473 "In vitro Mammalian Chromosome Aberration Test". The study comprised a preliminary toxicity test and six independent mutagenicity tests. The cells were exposed for 3 or 24 hours in the absence of exogenous metabolic activation (S9 mix) and 3 hours in the presence of S9 mix. In all tests the concurrent solvent and positive control were within the ranges determined from the historical solvent and positive control data. It was concluded that LAE did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of N^α-Lauroyl-L-arginine ethyl ester monohydrochloride (LAE) to induce chromosomal aberrations in human lymphocytes cultured in vitro.

 

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived from rat livers. Solvent and positive control cultures were also prepared. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

 

In order to assess the toxicity of LAE to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test

Without S9 mix - 3 h treatment, 17 h recovery: 50, 100 and 200 µg/ml

With S9 mix - 3 h treatment, 17 h recovery: 50, 100 and 200 µg/ml

Second test

Without S9 mix - 20 h continuous treatment: 50, 100 and 150 µg/ml

With S9 mix - 3 h treatment, 17 h recovery: 50, 100 and 150 µg/ml

In both the absence and presence of S9 mix, LAE caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control, in either test.

A quantitative analysis for polyploidy was made for cultures treated with the negative control and all dose levels of LAE used in the analysis for chromosomal aberrations. Statistically significant increases (P<0.01) in the proportion of polyploid cells were seen at the highest dose level, 200 ug/ml, in the first test in both the absence and the presence of S9 mix, and at the highest and intermediate dose levels, 150 and 100 µg/ml, in the second test in the presence of S9 mix.

 

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivityof the test system and the efficacy of the S9 mix.

 

It is concluded that LAE has shown no evidence of clastogenic activity in thisin vitro cytogenetic test system, under the experimental conditions described. Some evidence of polyploidy-inducing activitywas seen, but this was mainly at cytotoxic dose levels, and is unlikely to be of biological significance.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20, 1995 to August 30, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta-Somatic Cells: Detection of gene mutations in somatic cells in culture, 1983.

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: R0 (RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM
L-glutamine and 50 ug/mL gentamycin); R10p (R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v);
R30p (R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes.

- Type and identity of media:
RPMI 1640, obtained from a suitable supplier (Imperial Laboratories). This medium, supplemented with 0.1% Synperonic F68 (Serva), 0.011% sodium pyruvate (Sigma), 2 mM L-glutamine (Sigma), 50 /xg/ml gentamicin (Biological Industries) and buffered with 2 mg/ml sodium bicarbonate is referred to as ROp.
ROp supplemented with 10% HiDHS (supplied by Imperial Laboratories), referred to as RlOp, was used for general cell culture, eg when growing cells up from frozen stocks.
RlOp from which growing L5178Y cells had been removed was used as conditioned medium.
ROp in which the amount of Synperonic F68 had been reduced to 0.02% and supplemented with 30% HiDHS, referred to as R30p, formed the basis of the cloning medium. This was semi-solidified by the addition of Noble agar (Difco - in 0.9% saline) at a concentration of approximately 0.4%.
Selective medium consisted of cloning medium containing 4 /xg/ml TFT (Sigma).
- Properly maintained: yes

Additional strain / cell type characteristics:

other: Heterozygous at the thymidine kinase locus

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate derived from Aroclor 1254 induced rats (S-9 mix).

Test concentrations with justification for top dose:

Concentration range in the main test (without metabolic activation): -S9 mix
- Test 1 100, 150, 200, 220, 240, 260, 280 and 300 µg/mL
- Test 2 100, 150, 200, 220, 240, 260, 280 and 300 µg/mL

Concentration range in the main test (with metabolic activation): +S9 mix
- Test 1 100, 200, 300, 375, 400, 425, 450 and 500 µg/mL
- Test 2 100, 200, 300, 375, 400, 425, 450 and 500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: LAE was found to be soluble at approximately 500 mg/ml

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

other: 20-Methylcholanthrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:

PRELIMINARY TOXICITY TEST: in suspension;
A cell suspension was prepared at a population density of 1 x 10E(+6) cells/ml, in a 1 : 1 mixture of , R10p and conditioned media. The cell suspension was placed on a roller apparatus for approximately 30 minutes whilst the S-9 mix and the test compound solutions were prepared. 3 ml aliquots of this cell suspension were then dispensed into sterile universal tubes. 2 ml of ROp or S-9 mix was then added to each culture. One culture with and one without S-9 mix were prepared for each concentration of the test compound. The compound was diluted to provide serial concentrations which were incorporated into the cell suspensions. 50 /xl of test substance (at 100 times the desired final concentration) or solvent was added to each culture.

DURATION
- Exposure duration: 3 hours
- Selection time (if incubation with a selection agent): 48 h

SELECTION AGENT (mutation assays): Selective medium consisted of clonning medium containing 4 ug/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

MAIN TOXICITY TEST: in suspension;
Cell suspensions were prepared as described above with the following modifications: 12 ml aliquots of the cell suspension were dispensed into sterile 50 ml centrifuge tubes and 8 ml ROp or S-9 mix was then added; 200 µl of test substance, solvent or positive control were added to each culture. Two cultures, both in the presence and absence of S-9 mix, were used for each concentration of the test substance. At least four serial dilutions of the test compound were used, when possible at concentrations expected to span the LC80, and LC0 range. After addition of the test substance, solvent or positive control, all cultures were returned to the roller apparatus for 3 hours. Following the 3 hour treatment period, the cells were washed once before transferring to pre-gassed roller bottles in a final volume of 60 ml R10p. Cells were then returned to the roller apparatus for a further 48 hours to allow for expression of the mutant phenotype. The cultures were sampled as described above after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E(+5) cells/ml with RlOp. After the 48 hour expression period the cells were assessed for viability and mutant frequency by plating in semi-solid agar.

DURATION
- Exposure duration: 3 hours
- Selection time (if incubation with a selection agent): 48 h

SELECTION AGENT (mutation assays): Selective medium consisted of clonning medium containing 4 ug/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

Evaluation criteria:

The criteria for a positive response were:

An increase in mutant frequency in treated cultures of at least 100 relative to the concurrent control.
The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance.
Evidence of a dose relationship over at least two dose levels, in any increase in mutant frequency.
Demonstration of reproducibility in any increase in mutant frequency.
The observed increases in mutant frequency must lie outside the historical control range of 150 mutants per 106 survivors with a corresponding RTG of approximately 20%.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al (1989).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at the highest concentrations)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, treatment with 15 - 2000 µg/ml in the absence and presence of S-9 mix resulted in relative growth in suspension of 120 -1% and 109 - 0% respectively compared to the solvent controls. Concentrations used in the main test were based upon this data.

Conclusions:

The test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells according to OECD Guideline 476. It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro mammalian cell mutation assay.

Executive summary:

The test substance was tested for potential mutagenicity in the mouse lymphoma L5178Y cell mutation test according to OECD Guideline 476. This test involves detection of mutation of mouse lymphoma L5178Y cells in 96-well microtitre plates. The cells are heterozygous at the thymidine kinase (tk) locus (TK^/-), and forward mutation (TK^-/-) is detected by the ability of these cells to divide and form colonies in the presence of trifluorothymidine (TFT), a toxic analogue of thymidine.

Two independent tests in the absence of exogenous metabolic activation and two independent tests in the presence of S-9 mix were carried out.

In the preliminary toxicity test, treatment with 15 - 2000 µg/ml in the absence and presence of S-9 mix resulted in relative growth in suspension of 120 -1% and 109 - 0% respectively compared to the solvent controls. Concentrations used in the main test were based upon this data.

 

In the absence of S-9 mix treatment of cells with 100 - 300 µg/ml in Test 1and Test 2 resulted in mean cell growths in suspension of 96-21% and 94-1 % respectively. Cultures treated with 100, 200, 280 and 300 µg/ml in Test 1 and 150, 220, 240 and 280 µg/ml in Test 2 were cloned in soft agar to permit measurement of the levels of viability and induced mutation. The resulting RTGs were 86 - 24% in Test 1 and 77 - 35% in Test 2 relative to the controls.

 

No significant increases in mutant frequency were observed after treatment with the test substance in either test. EMS, the positive control, induced highly significant increases in mutant frequency in both tests.

In the presence of S-9 mix treatment of cells with 100 - 500 µg/ml in Test 1 and Test 2 resulted in mean cell growths in suspension of 82 -2% and 90 -2% respectively. Cultures treated with 200, 300, 400, 425 and 450 µg/ml in Test 1 and 200, 300, 400 and 450 µg/ml in Test 2 were cloned in soft agar to permit measurement of the levels of viability and induced mutation. The resulting RTGs were 70 -8% in Test 1 and 83 - 32% in Test 2 relative to the controls.

 

No significant increases in mutant frequency were observed after treatment with the test substance in either test. In the second test a higher than normal control mutant frequency was observed but this was not considered to adversely affect the results obtained. 20-Methylcholanthrene, the positive control, induced highly significant increases in mutant frequency in both tests.

 

Toxicity was observed after treatment with the test substance in both tests in the absence and the presence of S-9 mix.

It was, then, concluded that the test substance did not demonstrate mutagenic potentialin thisin vitrocell mutation assay, under the experimental conditions described.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 25, 2001 to June 13, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Department of Health Report on Health and Social Subjects No. 35. Guidelines for the testing of chemicals for mutagenicity (1989).

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human blood was collected aseptically from healthy male donors.

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Healthy male donors
- Whether whole blood or separated lymphocytes were used if applicable: Separated by centrifugation.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 +20% foetal calf serum + phytohaemaglglutinin (175 µg/mL). Incubation aat 5 % CO2
- Properly maintained: yes

Cytokinesis block (if used):

Colchicine

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate derived from Aroclor 1254 induced rats (S-9 mix).

Test concentrations with justification for top dose:

FIRST TEST
Without S-9 mix, 18 hour harvest: 125, 250 and 500 µg/ml.
With S-9 mix, 18 hour harvest: 125, 250 and 500 µg/ml.

SECOND TEST
Without S-9 mix, 18 hour harvest: 125,250 and 500 µg/ml.
With S-9 mix, 18 hour harvest: 700, 800 and 1000 µg/ml.
Without S-9 mix, 32 hour harvest: 500 µg/ml.
With S-9 mix, 32 hour harvest: 1000 µg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

FIRST TEST:
METHOD OF APPLICATION: preincubation;
After 48 hours, 50 µl aliquots of the test substance were added to one set of duplicate cultures to give final concentrations of 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/ml. Water, the solvent control, in 50 µl aliquots, was added to four cultures. Ethyl methanesulphonate, at final concentrations of 500 and 750 µg/ml, was added to duplicate cultures. 1.25 ml S-9 mix was added to each culture in a second set, followed by 62.5 µl aliquots of the various dilutions of the test substance, giving the same series of final concentrations as above. Water (62.5 µl) was added to four cultures. Cyclophosphamide was added to duplicate cultures at final concentrations of 10 and 15 µg/ml. Three hours after dosing, the cultures containing the S-9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 15 hours. The cultures treated in the absence of S-9 mix were incubated for 18 hours.

DURATION
- Exposure duration: 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 15 hours (+S-9) and 18 hours (-S-9)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: One hundred metaphase figures were examined, from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; The proportion of mitotic cells per 1000 cells in each culture was recorded.

SECOND TEST:
METHOD OF APPLICATION: preincubation;
Cultures were initiated and maintained as previously described. Cultures to be harvested 32 hours after initiation of treatment were treated with 125, 250, 500, 1000 and 2000 µg/ml of the test substance in the absence of S-9 mix and with 250, 500 and 1000 µg/ml in the presence of S-9 mix. Concentrations of the test substance dosed for the second 18 hour test were 125, 250, 500, 750, 1000, 1500 and 2000 µg/ml in the absence of S-9 mix and 125, 250, 500, 600, 700, 800 and 1000 µg/ml in its presence. Duplicate cultures were used for each treatment and four cultures were treated with the solvent control. For the 32 hour harvest ethyl methanesulphonate was added to duplicate cultures at final concentrations of 250 and 500 µg/ml, and cyclophosphamide was added to duplicate cultures at final concentrations of 2.5 and 5 µg/ml. Three hours after dosing, the cultures containing S-9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 15 or 29 h. Cultures treated in the absence of S-9 mix were incubated for 18 or 32 h. All cultures were treated with colchicine at a final concentration of 0.25 µg/ml two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were then examined microscopically. For the 32 hour harvest only one dose level was selected for analysis. Where possible, this was the same as the highest dose level chosen from the 18 hour harvest.

DURATION
- Exposure duration: 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18/32 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: One hundred metaphase figures were examined, from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; The proportion of mitotic cells per 1000 cells in each culture was recorded.

Key result

Species / strain:

lymphocytes: Separated from human blood collected from healthy male donors

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Separated from human blood collected from healthy male donors

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at the highest concentration)

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

According to OECD 473, the test substance was tested for mutagenic potential in an in vitro mammalian cell mutation assay in an vitro Mammalian Chromosome Aberration Test. Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Metaphase figures could be examined for chromosomal damage. After observations tt was concluded that the test substance showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in human lymphocytes cultured in vitro.

 

The study was conducted in compliance with the OECD Guidelines for Testing of Chemicals No.473 "Genetic Toxicology: In vitro Mammalian Cytogenetic Test".

 

Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treatment time, cell division was arrested using colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage.

 

In order to assess the toxicity of the test substance to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

 

FIRST TEST

Without S-9 mix, 18 hour harvest: 125, 250 and 500 µg/ml.

With S-9 mix, 18 hour harvest: 125, 250 and 500 µg/ml.

SECOND TEST

Without S-9 mix, 18 hour harvest: 125, 250 and 500 µg/ml.

With S-9 mix, 18 hour harvest: 700, 800 and 1000 µg/ml.

Without S-9 mix, 32 hour harvest: 500 µg/ml.

With S-9 mix, 32 hour harvest: 1000 µg/ml.

The test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level, in either the absence or presence of S-9 mix, when compared with the solvent control.

 

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells.

 

It is concluded that the test substance showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, LAE is not genotoxic and it is not classified for genetic toxicity according to CLP Regulation.